This invention relates to the use of ribozymes as inhibitors of human immunodeficiency virus (HIV) replication, and in particular, the inhibition of HIV-1 replication. See e.g., Draper et al., PCT/WO93/23569 hereby incorporated by reference.
Acquired immunodeficiency syndrome (AIDS) is thought to be caused by infection with the virus HIV-1. At present, it is treated by administration of the drug azidothymidine (AZT), which is thought to slow the progress of, but not cure, the disease. AZT resistant strains of HIV-1 are found to develop after a year of treatment. In some patients AZT has limited efficacy and may be found intolerable. More recently, drugs such as dideoxyinosine (DDI) and dideoxycytidine (DDC) have been tested as treatments for AIDS. None of these compounds reduce the viral load in patients, but they do treat the disease symptoms.
The following is a discussion of relevant art, none of which is admitted to be prior art to the pending claims. Rossi et al., 8 Aids Research and Human Retroviruses 183, 1992, provide a review of the use of ribozymes as anti-HIV-1 therapeutic agents. They state:
An emerging strategy in the treatment of viral infections is the use of antisense DNA or RNA to pair with, and block expression of viral transcripts. RNA, in addition to being an informational molecule, can also possess enzymatic activity. Thus, by combining anti-sense and enzymatic functions into a single transcript, it is now possible to design catalytic RNAs, or ribozymes, which can specifically pair with virtually any viral RNA, and cleave the phosphodiester backbone at a specified location, thereby functionally inactivating the viral RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. There are several different catalytic motifs which possess enzymatic activity, and each one of these can be incorporated into an enzymatic antisense with site-specific cleavage capabilities.
Rossi et al. also state that studies have demonstrated that a hammerhead ribozyme targeted to the gag gene RNA in the vicinity of the translational initiation codon is capable of specifically cleaving that target in a complex milieux of total cellular RNA. With reference to identification of ribozyme targets in HIV-1 they state that mRNAs for the two regulatory proteins tat and rev are clearly targets of choice, and that they are examining potential ribozyme cleavage sites in the tat mRNA, as well as in the exon shared by tat and rev. In addition, they state:
A rational approach to the problem of target selection involves the following criteria. First, one should select a functionally important target, such as tat, rev, int, psi (packaging site), or the tRNA.sup.lys priming site. Once a gene or target region has been decided upon, the nucleotide sequence should be assessed for strong conservation of sequence among the various isolates. Within these conserved regions, the potential cleavage sites, preferably GUC or GUA (others will suffice, but appear to be less efficiently cleaved) should be chosen. The region should be examined for potential secondary structures, and then the most promising sites chosen. Finally, before testing the ribozyme in cell culture, it is advisable to carry out a series of in vitro cleavage reactions (preferably kinetic analyses) using long (at least 100 nucleotides in length) substrates to verify that the chosen sites are truly structurally favorable for cleavage. [Citation omitted.]
Rossi et al. further state that a target which deserves further consideration and testing as a potential ribozyme cleavage site is the viral packaging signal or psi sequence.
Sioud and Drlica, 88 Proc. Natl. Acad. Sci. USA 7303, 1991 describe ribozymes designed to cleave the integrase gene of HIV. They state that when the ribozyme is transcribed from a plasmid in E. coli it leads to destruction of the integrase RNA and complete blockage of integrase protein synthesis. They state that the HIV-1 integrase gene may be a useful target for therapeutic ribozymes.
Heidenreich and Eckstein, 267 Journal of Biological Chemistry 1904, 1992, describe three ribozymes targeted to different sites on the long terminal repeat (LTR) RNA of HIV-1. They also describe the influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA, including 2'-Fluorocytidine substitutions and phosphorothioate internucleotidic linkages.
Weerasinghe et al., 65 Journal of Virology 5531, 1991, describe ribozymes designed against a conserved region within the 5' leader sequence of HIV-1 RNA.
Chang et al., 2 Clinical Biotechnolocy 23, 1990, describe ribozymes designed to target two different sites in the HIV-1 gag gene, and a single site in the viral 5'-LTR region.
Lorentzen et al., 5 Virus Genes 17, 1991, describe a ribozyme targeted to the virion infectivity factor (vif) of HIV-1.
Sarver et al., 247 Science 1222, 1990, describe ribozymes in the hammerhead family targeted to HIV-1 gag transcripts. They state that cells challenged with HIV-1 showed a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, and that the reduction in gag RNA was reflected by a reduction in antigen p24 levels. They state that the results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.
Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100, filed Sep. 20, 1988, describe hairpin ribozymes, and provides an example of such a ribozyme apparently specific to the gag gene of HIV-1. Hampel and Tritz, 28 Biochemistry 4929, 1989 and Hampel et al., 18 Nucleic Acids Research 299, 1990 also describe hairpin catalytic RNA models and state that one target site is the tat gene in HIV-1.
Goldberg et al., WO 91/04319 and Robertson and Goldberg WO 91/04324, describe ribozymes expressed within a hepatitis delta vector and state that the genome of the delta virus may carry a ribozyme against the env or gag mRNA of HIV. Rossi et al., WO 91/03162, describe chimeric DNA-RNA catalytic sequences used to cleave HIV-1 gag transcript or the 5' LTR splice site.
Ojwang et al., 89 Proc. Natl. Acad. Sci. USA 10, 802, 1992 and Yu et al., 90 Proc. Natl. Acad. Sci. USA 6340, 1993 describe a hairpin ribozyme allegedly able to inhibit HIV-1 expression. Joseph and Burke 268 J. Biol. Chem. 24, 515 1993, describe optimization of an anti-HIV hairpin ribozyme. Dropulic et al., 66 J. Virology 1432, 1992 describe a U5 ribozyme which cleaves at nucleoside +115 in HIV-1 RNA.
Other related art includes Rossi et al., U.S. Pat. Nos. 5,144,019 and 5,149,796; Altman et al., U.S. Pat. No. 5,168,053; Zaia et al., 660 Ann. N.Y. Acad. Sci. 95, 1992; Guatelli et al., 16E J. Cell Biochem. 79, 1992; Jeang et al., 267 J. Biol. Chem. 17891, 1992; Dropulic et al., 66 J. Virol. 1432, 1992; Lisziewicz et al., International Publication WO 91/10453; International Publication WO 91/15500; Rossi et al., 14A J. Cell Biochem. D428, 1990; and "The Papovaviridae", Ed. Salzman et al., Vol. 2, The Viruses, Plenum Press, New York 1987.
Ribozymes are RNA molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage has been achieved in vitro. Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989.
Ribozymes act by first binding to a target RNA. Such binding occurs through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA which acts to cleave the target RNA. Thus, the ribozyme first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After a ribozyme has bound and cleaved its RNA target it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.